Colicin

Colicins are a type of bacteriocin - peptide and protein antibiotics released by bacteria to kill other bacteria of the same species, in order to provide a competitive advantage for nutrient acquisition. Bacteriocins are named after their species of origin; colicins are so-called because they are produced by E. Coli. Because of their narrow killing spectrum which focuses primarily on the species which has made the peptide (or occasionally closely related species ), bacteriocins are important in microbial biodiversity and the stable co-existence of the bacterial populations.

Colicin peptides are plasmid-encoded. The peptide is released by the cell into the area surrounding it, and then parasitises proteins present in the host cell membrane to translocate across into the host cell. Many protein-protein interactions are involved in the cell entry, and the main system is involved in the grouping of colicins into two families: Group A colicins use the Tol system to enter the host cell, and Group B use the Ton system. Once inside the host cell, the cell killing follows 1st order kinetics - ie one molecule is theoretically sufficient to kill the cell.

The structure of all colicins, of which over 20 have been identified, follows a 3 domain design: At the N terminus is the Translocation domain (T-): Residues 1 to ~ 190 in ColIa. The Receptor binding domain is at the centre of the peptide (R-): Residues ~190 to 451 in ColIa. The C terminus contains the Cytotoxic domain (C-): Residues 452 to 626 in ColIa.

Synthesis, Production and Release
Synthesis of many colicins is repressed by the LexA protein, which is part of the SOS regulon.



Targeting and Receptors
Colicins vary significantly in the receptors that they target to initiate their uptake. The majority of the group A colicins use the BtuB receptor, which is present on E. coli as a vitamin B12 uptake receptor. Once bound to the receptor, the coiled-coil receptor binding domain unfolds, in an essential step that removes the immunity protein and triggers translocation. Other colicins use other receptors - generally involved in the uptake of small metabolite growth factors.

Colicin Uptake
Colicins are divided into two groups depending on the method of uptake which they target. Group A colicins use the Tol system to bind to and enter the target cell, and group B use the Ton system. The Tol system consists of 5 proteins - TolA, TolB, TolR, TolQ and Pal, and group A proteins using this often recruit a second co-receptor involved in translocation, usually OmpF or TolC, but could be OmpC and PhoE. The Ton system consists of TonB, ExbB and ExbD, and no known co-receptor is utilised in translocation .It could be possible that Ton-dependent colicins are indiscriminate in use of coreceptors, or that the colicins move down the outside wall of a &beta; barrel protein. It is known that colicins do unfold during translocation, but the peptides resulting from this exceed the diameter of pores formed by any of the molecules mentioned above. However, while unfolding does occur, this is not induced by receptor binding in either Tol or Ton dependent colicins.

Understanding how the colicins can cross the membrane is highly important, as if this could be targeted and exploited it could be useful for novel therapeutic agents. It is also estimated that a single colicin molecule is sufficient to kill the bacterial cell, following first order kinetics.

Killing Activities
Colicins kill their target cell through a variety of different methods. The main killing activities are carried out through Pore Formation, DNase Activity and 16s rRNase activity, and some colicins also exhibit tRNase activity.

The killing activities carried out by colicins could be used medicinally as an alternative to antibiotics in the case where the specific strain of E. coli can be identified, and as potential natural replacements for food preservatives.

Table taken from except where indicated.

Directed evolution and Colicin7/Immunity-proteins complexes
 Iterative rounds of random mutagenesis and selection of immunity protein 9 (colored yellow) toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation variants R12-2 (3gkl; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and R12-13 (3gjn; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved.

Structural alignment of the immunity protein 9 (Im9, 1bxi, colored yellow), evolved variant R12-2 (lime), and immunity protein 7 (Im7, 7cei, colored blue) reveals their structural identity. However, when the immunity proteins-bound colicins within their complexes were aligned, they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 (1bxi) there are Tyr54 and Tyr55, while in the Im7 (7cei) Tyr55 and Tyr56 are homologous to them. The most prominent differences are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and evolved variant R12-2 (lime, labeled in black). This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28.

Comparison of the different Im-colicin complexes reveals changes in the binding configuration of the evolved variants which increase affinity toward ColE7 by re-aligning pre-existing Im9 residues. Glu30 of Im9 (1bxi, colored yellow) forms double salt bridge with Arg54 of ColE9 (orange), whereas Asp51 have not direct side chain–side chain interactions. Asp31 of Im7 (blue) (corresponding to Im9 Glu30) is involved in <scene name='3gkl/Active_site/6'>cluster of salt bridge bonds to <font color='darkmagenta'>Arg520 and Lys525 of ColE7 (darkmagenta), while <font color='blue'>Asp52 of Im7 (corresponding to Im9 Asp51) is within hydrogen bond distance to <font color='darkmagenta'>Thr531 and Arg530 of ColE7. <font color='lime'>Glu30 in the variant R12-2 (lime) is shifted and forms a <scene name='3gkl/Active_site/8'>double salt bridge to <font color='magenta'>Arg520 of ColE7 (magenta). <font color='lime'>Asp51 is within hydrogen bond distance to <font color='magenta'>Thr531 of ColE7. However, the side chains of <font color='magenta'>Lys525 and Arg530, which are very important in salt bridge contacts with Glu30 and Asp51, respectively, in the structure of the ColE7–Im7 complex have a different conformation that eliminates these contacts in evolved variant R12-2.

In the <scene name='3gkl/Mut/2'>Im9 <font color='magenta'>Val37 (colored magenta) forms stabilizing hydrogen bond with Leu33. In the <scene name='3gkl/Mut/3'>evolved variant R12-2, <font color='darkmagenta'>Ile37 (colored darkmagenta) interacts with two additional residues, Tyr54 and Ser50. Moreover, <font color='darkmagenta'>Ile37 also forms additional hydrogen bond with Gly41 and can thereby have enabled the appearance of the selectivity mutation E41G.

In contrast to the <font color='lime'>evolved variant R12-2 (3gkl), the <font color='cyan'>evolved variant R12-13 (3gjn) carries the <scene name='3gkl/Align/10'>Tyr55Trp mutation in the conserved region. Both <font color='lime'>Tyr55 in R12-2 and <font color='cyan'>Trp55 in R12-13 could sustain the hydrophobic core and create a <scene name='3gkl/Align/11'>hydrogen bond to Lys528 backbone (3gkl colicin residues are colored in <font color='magenta'>magenta, 3gjn colicin residues are colored <font color='blueviolet'>blueviolet ). However, the additional bulkiness of the Trp contributes in expanding its <scene name='3gkl/Align/9'>hydrophobic interactions to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530.

The <scene name='3gkl/Ali/1'>overall conformation of the two evolved variants <font color='lime'>R12-2 (3gkl) and <font color='cyan'>R12-13 (3gjn) is very similar. The variant <font color='lime'>R12-2 carries <scene name='3gkl/Ali/2'>mutation E41G. In the bound wildtype Im9 (yellow) Glu41 makes a <scene name='3gkl/Ali/3'>salt bridge with the <font color='orange'>ColE9’s Lys97 (1bxi). While in the <font color='blueviolet'>R12-13 /<font color='cyan'>ColE7 complex the <font color='blueviolet'>closest ColE7 residues <scene name='3gkl/Ali/4'>contacting <font color='cyan'>R12-13 Glu41 are <font color='blueviolet'>Thr531 (3.37Å) and Lys528 (8.85Å) (3gjn). In the <font color='lime'>R12-2 /<font color='magenta'>ColE7 complex the <scene name='3gkl/Ali/5'>closest <font color='magenta'>ColE7 residue to <font color='lime'>R12-2 Gly41 is <font color='magenta'>Thr531 (9.48Å) (3gkl).

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3D structure of Colicin
Update June 2011

Colicin-A
3iax – CfColA translocation domain + EcTolB – Citrobacter freundii

1col – EcColA pore-forming domain

Colicin-B
1rh1 - EcColB

Colicin-D
1tfk, 1tfo, 1v74 - EcColD catalytic domain + EcColD immunity protein

Colicin-E1
2i88 – EcColE1 channel-forming domain

Colicin-E2
2ysu – EcColE2 receptor-binding domain + BtuB

Colicin-E3
2xfz, 2xg1 – EcColE3 cytotoxic domain (mutant) + Tt30S ribosome – Thermus thermophilus

2zld - EcColE3 cytotoxic domain + outer membrane protein F

1jch – EcColE3 + EcColE3 immunity protein

2b5u – EcColE3 (mutant) + EcColE3 immunity protein

1e44 – EcColE3 nuclease domain + EcColE3 immunity protein

1ujw – EcColE3 receptor-binding domain + BtuB

Colicin-E5
2djh – EcColE5 C-terminal domain

2a8k – EcColE5 catalytic domain

2dfx – EcColE5 C-terminal domain + EcColE5 immunity protein

2fhz – EcColE5 residues 74-180 + EcColE5 immunity protein

Colicin-E7
1unk - EcColE7

2axc - EcColE7 translocation domain

1m08 - EcColE7 nuclease domain

3fbd, 1zns – EcColE7 (mutant) + DNA

1pt3 - EcColE7 nuclease domain + DNA

1mz8, 7cei - EcColE7 nuclease domain + EcColE7 immunity protein

3gjn - EcColE7 nuclease domain + EcColE9 immunity protein (mutant)

3gkl - EcColE7 nuclease domain (mutant) + EcColE9 immunity protein (mutant)

2jaz, 2jb0, 2jbg, 1znv - EcColE7 nuclease domain (mutant) + EcColE7 immunity protein

2erh, 1ujz - EcColE7 (mutant) + EcColE7 immunity protein (mutant)

Colicin-E9
1fsj - EcColE9 DNase domain

1v13 - EcColE9 DNase domain (mutant)

1v14, 1v15 - EcColE9 DNase domain (mutant) + DNA

2wpt – EcColE9 (mutant) + EcColE2 immunity protein

2k5x, 1emv, 1bxi – EcColE9 DNase domain + EcColE9 immunity protein

2vln, 2vlo, 2vlp, 2vlq - EcColE9 DNase domain (mutant) + EcColE9 immunity protein

2gze, 2gzg, 2gzi, 2gzj, 2gzf, 2gyk, 1fr2 - EcColE9 DNase domain + EcColE9 immunity protein (mutant)

2ivz - EcColE9 T domain + TolB

3o0e - EcColE9 fragment + outer membrane porin 1A

Colicin-Ia
1cii - EcColIa

2hdi – EcColIA R domain + ColI receptor

Colicin-M
2xmx, 3da3, 3da4 – EcColM

2xtq, 2xtr – EcColM (mutant)

Colicin-N
1a87 – EcColN receptor-binding domain

Colicin-S4
3few – EcColS4